Human manganese superoxide dismutase DNA, its expression and method of recovering human manganese superoxide dismutase

ABSTRACT

A double-stranded cDNA molecule which includes DNA encoding human manganese superoxide dismutase has been created. The sequence of one strand of a double-stranded DNA molecule which encodes human manganese superoxide dismutase has been discovered. Such molecules may be introduced in procaryotic, e.g., bacterial, or eukaryotic, e.g., yeast or mammalian, cells and the resulting cells cultured or grown under suitable conditions so as to produce human manganese superoxide dismutase or analogs thereof which may then be recovered. By this invention, human MnSOD gene fragments from various plasmids may be ligated to yield a complete genomic human MnSOD gene fragment. Human MnSOD or analogs thereof may be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of isolated organs, or treat inflammations. 
     The invention also concerns a method of producing enzymatically active human manganese superoxide dismutase or an analog thereof in a bacterial cell which contains and is capable of expressing a DNA sequence encoding the superoxide dismutase by maintaining the bacterial cell under suitable conditions and in a suitable production medium. The production medium is supplemented with an amount of Mn ++  so that the concentration of Mn ++  in the medium is greater than about 2 ppm. Genomic MnSOD DNA should also be capable of expression in eucaryotic cells under suitable conditions. 
     This invention also concerns a method of recovering purified enzymatically active manganese superoxide dismutase from bacterial cells. It should also be possible to recover manganese SOD from genomic MnSOD DNA expressed in eucaryotic cells using similar methods.

This application is a continuation of U.S. Ser. No. 08/370,461, filedJan. 9, 1995, now U.S. Pat. No. 5,540,911, issued Jul. 30, 1996; whichis a continuation of U.S. Ser. No. 08/120,951, filed Sep. 14, 1993, nowabandoned; which is a divisional of U.S. Ser. No. 07/912,213, filed Jul.10, 1992, now U.S. Pat. No. 5,270,195, issued Dec. 14, 1993; which is acontinuation of U.S. Ser. No. 07/453,057, filed Dec. 13, 1989, nowabandoned; which is a continuation of U.S. Ser. No. 07/032,734, filedMar. 27, 1987, now abandoned; which was a continuation-in-part of Ser.No. 06/907,051, filed Sep. 12, 1986, now abandoned; which was acontinuation-in-part of Ser. No. 06/801,090, filed Nov. 22, 1985, nowabandoned.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byarabic numerals within parentheses. Full citations for these referencesmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of art as known to those skilled therein as ofthe date of the invention described and claimed herein.

Superoxide dismutase (SOD) and the phenomenon of oxygen free radicals(O₂) was discovered in 1968 by McCord and Fridovich (1). Superoxideradicals and other highly reactive oxygen species are produced in everyrespiring cell as by-products of oxidative damage to a wide variety ofmacromolecules and cellular components (for review see 2,3). A group ofmetalloproteins known as superoxide dismutases catalyze theoxidation-reduction reaction 20₂+₂H⁺→H₂O₂+O₂ and thus provide a defensemechanism against oxygen toxicity.

There are several known forms of SOD containing different metals anddifferent proteins. Metals present in SOD include iron, manganese,copper and zinc. All of the known forms of SOD catalyze the samereaction. These enzymes are found in several evolutionary groups.Superoxide dismutases containing iron are found primarily in prokaryoticcells. Superoxide dismutases containing copper and zinc has been foundin virtually all eukaryotic organisms (4). Superoxide dismutasescontaining manganese have been found in organisms ranging frommicroorganisms to man.

Since every biological macromolecule can serve as a target for thedamaging action of the abundant superoxide radical, interest has evolvedin the therapeutic potential of SOD. The scientific literature suggeststhat SOD may be useful in a wide range of clinical applications. Theseinclude prevention of oncogenesis and of tumor promotion, and reductionof the cytotoxic and cardiotoxic effects of anticancer drugs (10),protection of ischemic tissues (12) and protection of spermatozoa (13).In addition, there is interest in studying the effect of SOD on theaging process (14).

The exploration of the therapeutic potential of human SOD has beenlimited mainly due to its limited availability.

Superoxide dismutase is also of interest because of itsanti-inflammatory properties (11). Bovine-derived superoxide dismutase(orgotein) has been recognized to possess anti-inflammatory propertiesand is currently marketed in parts of Europe as a human pharmaceutical.

It is also sold in the United States as a veterinary product,particularly for the treatment of inflamed tendons in horses. However,supplies of orgotein are limited. Prior techniques involving recoveryfrom bovine or other animal cells have serious limitations and theorgotein so obtained may produce allergic reactions in humans because ofits non-human origin.

Copper zinc superoxide dismutase (CuZn SOD) is the most studied and bestcharacterized of the various forms of superoxide dismutase.

Human CuZn SOD is a dimeric metallic-protein composed of identicalnon-covalently linked subunits, each having a molecular weight of 16,000daltons and containing one atom of copper and one of zinc (5). Eachsubunit is composed of 153 amino acids whose sequence has beenestablished (6,7).

The cDNA encoding human CuZn superoxide dismutase has been cloned (8).The complete sequence of the cloned DNA has also been determined (9).Moreover, expression vectors containing DNA encoding superoxidedismutase for the production and recovery of superoxide dismutase inbacteria have been described (24,25). The expression of a superoxidedismutase DNA and the production of SOD in yeast has also been disclosed(26).

Recently, the CuZn SOD gene locus on human chromosome 21 has beencharacterized (27) and recent developments relating to CuZn superoxidedismutase have been summarized (28).

Much less is known about manganese superoxide dismutase (MnSOD). TheMnSOD of E. coli K-12 has recently been cloned and mapped (22). Barra etal. disclose a 196 amino acid sequence for the MnSOD polypeptideisolated from human liver cells (19). Prior art disclosures differ,however, concerning the structure of the MnSOD molecule, particularlywhether it has two or four identical polypeptide subunits (19,23). It isclear, however, that the MnSOD polypeptide and the CuZn SOD polypeptideare not homologous (19). The amino acid sequence homologies of MnSODsand FeSOD from various sources have also been compared (18).

Baret et al. disclose in a rat model that the half life of human MnSODis substantially longer than the half-life of human copper SOD; theyalso disclose that in the rat model, human MnSOD and rat copper SOD arenot effective as anti-inflammatory agents whereas bovine copper SOD andhuman copper SOD are fully active (20).

McCord et al. disclose that naturally occurring human manganesesuperoxide dismutase protects human phagocytosing polymorphonuclear(PMN) leukocytes from superoxide free radicals better than bovine orporcine CuZn superoxide dismutase in “in vitro” tests (21).

The present invention concerns the preparation of a cDNA moleculeencoding the human manganese superoxide dismutase polypeptide or ananalog or mutant thereof. It is also directed to inserting this cDNAinto efficient bacterial expression vectors, to producing human MnSODpolypeptide, analog, mutant and enzyme in bacteria, to recovering thebacterially produced human MnSOD polypeptide, analog, mutant or enzyme.This invention is also directed to the human MnSOD polypeptides,analogs, or mutants thereof so recovered and their uses.

This invention further provides a method for producing enzymaticallyactive human MnSOD in bacteria, as well as a method for recovering andpurifying such enzymatically active human MnSOD.

The present invention also relates to a DNA molecule encoding the humanMnSOD gene. It is also directed to inserting the DNA into mammaliancells to produce MnSOD polypeptide, analog, mutant and enzyme.

The present invention also relates to using human manganese superoxidedismutase or analogs or mutants thereof to catalyze the reduction ofsuperoxide radicals to hydrogen peroxide and molecular oxygen. Inparticular, the present invention concerns using bacterially producedMnSOD or analogs or mutants thereof to reduce reperfusion injuryfollowing ischemia and prolong the survival period of excised isolatedorgans. It also concerns the use of bacterially produced MnSOD oranalogs thereof to treat inflammations.

SUMMARY OF THE INVENTION

A DNA molecule which includes cDNA encoding the human manganesesuperoxide dismutase polypeptide or analog or mutant thereof has beenisolated from a human T-cell library. The nucleotide sequence of adouble-stranded DNA molecule which encodes human manganese superoxidedismutase polypeptide or analog or mutant thereof has been discovered.The sequence of one strand encoding the polypeptide or analog thereof isshown in FIG. 1 from nucleotide 115 downstream to nucleotide 708inclusive. Other sequences encoding the analog or mutant may besubstantially similar to the strand encoding the polypeptide. Thenucleotide sequence of one strand of a double stranded DNA moleculewhich encodes a twenty-four (24) amino acid prepeptide is also shown inFIG. 1, from nucleotides number 43 through 114, inclusive.

The double-stranded cDNA molecule or any other double-stranded DNAmolecule which contains a nucleotide strand having the sequence encodingthe human manganese superoxide dismutase polypeptide or analog or mutantthereof may be incorporated into a cloning vehicle such as a plasmid orvirus. Either DNA molecule may be introduced into a cell, eitherprocaryotic, e.g., bacterial, or eukaryotic, e.g., yeast or mammalian,using known methods, including but not limited to methods involvingcloning vehicles containing either molecule.

Preferably the cDNA or DNA encoding the human manganese superoxidedismutase polypeptide or analog or mutant thereof is incorporated into aplasmid, e.g., pMSE-4 or pMSΔRB4, and then introduced into a suitablehost cell where the DNA can be expressed and the human manganesesuperoxide dismutase (hMnSOD) polypeptide or analog or mutant thereofproduced. Preferred host cells include Escherichia coli, in particularE. coli A4255 and E. coli A1645. The plasmid pMSE-4 in E. coli strainA4255 has been deposited with the American Type Culture Collection underATCC Accession No. 53250. The plasmid pMS RB4 may be obtained as shownin FIG. 4 and described in the Description of the Figures.

Cells into which such DNA molecules have been introduced may be culturedor grown in accordance with methods known to those skilled in the artunder suitable conditions permitting transcription of the DNA into mRNAand expression of the mRNA as protein. The resulting manganesesuperoxide dismutase protein may then be recovered.

Veterinary and pharmaceutical compositions containing human MnSOD oranalogs or mutants thereof and suitable carriers may also be prepared.This human manganese superoxide dismutase or analogs or mutants may beused to catalyze the following reaction:

20₂+2H⁺→H₂O₂+O₂

and thereby reduce cell injury caused by superoxide radicals.

More particularly, these enzymes or analogs or mutants thereof may beused to reduce injury caused by reperfusion following ischemia, increasethe survival time of excised isolated organs, or treat inflammations.

This invention is directed to a method of producing enzymatically activehuman manganese superoxide dismutase or an analog or mutant thereof in abacterial cell. The bacterial cell contains and is capable of expressinga DNA sequence encoding the manganese superoxide dismutase or analog ormutant thereof. The method comprises maintaining the bacterial cellunder suitable conditions and in a suitable production medium. Theproduction medium is supplemented with an amount of Mn⁺⁺ so that theconcentration of Mn⁺⁺ available to the cell in the medium is greaterthan about 2 ppm.

In a preferred embodiment of the invention the bacterial cell is anEscherichia coli cell containing a plasmid which contains a DNA sequenceencoding for the human manganese superoxide dismutase polypeptide e.g.pMSE-4 or pMSΔRB4 in E. coli strain A4255. The concentration of Mn⁺⁺ inthe production medium ranges from about 50 to about 1500 ppm, withconcentrations of 150 and 750 ppm being preferred.

This invention also concerns a method of recovering manganese superoxidedismutase or analog thereof from bacterial cells which contain the same.The cells are first treated to recover a protein fraction containingproteins present in the cells including human manganese superoxidedismutase or analog or mutant thereof and then the protein fraction istreated to recover human manganese superoxide dismutase or analog ormutant thereof. In a preferred embodiment of the invention, the cellsare first treated to separate soluble proteins from insoluble proteinsand cell wall debris and the soluble proteins are recovered. The solubleproteins are then treated to separate, e.g. precipitate, a fraction ofthe soluble proteins containing the hMnSOD or analog or mutant thereofand the fraction containing the hMnSOD or analog or mutant is recovered.The recovered fraction of soluble proteins is then treated to separatelyrecover the human manganese superoxide dismutase or analog thereof.

A more preferred embodiment of the invention concerns a method ofrecovering human manganese superoxide dismutase or analog or mutantthereof from bacterial cells which contain human manganese superoxidedismutase or analog or mutant thereof. The method involves firstisolating the bacterial cells from the production medium and suspendingthem in suitable solution having a pH of about 7.0 to 8.0. The cells arethen disrupted and centrifuged and the resulting supernatant is heatedfor about 30 to 120 minutes at a temperature between 55 and 65° C.,preferably for 45-75 minutes at 58-62° C. and more preferably for 1 hourat 60° C. and then cooled to below 10° C., preferably to 4° C. Anyprecipitate which forms is to be removed e.g. by centrifugation, and thecooled supernatant is dialyzed against an appropriate buffer e.g. 2 mMpotassium phosphate buffer having a pH of about 7.8. Preferably, thedialysis is by ultrafiltration using a filtration membrane smaller than30K. Simultaneously with or after dialysis the cooled supernatantoptionally may be concentrated to an appropriate convenient volume e.g.0.03 of its original volume. The retentate is then eluted on an anionexchange chromatography column with an appropriate buffered solutione.g. a solution of at least 20 mM potassium phosphate buffer having a pHof about 7.8. The fractions of eluent containing superoxide dismutaseare collected, pooled and dialyzed against about 40 mM potassiumacetate, pH 5.5. The dialyzed pooled fractions are then eluted through acation exchange chromatography column having a linear gradient of about40 to about 200 mM potassium acetate and a pH of 5.5. The peak fractionscontaining the superoxide dismutase are collected and pooled. Optionallythe pooled peak fractions may then be dialyzed against an appropriatesolution e.g. water or a buffer solution of about 10 mM potassiumphosphate buffer having a pH of about 7.8.

The invention also concerns purified enzymatically active humanmanganese superoxide dismutase or analogs thereof e.g. met-hMnSOD, ormutants produced by the methods of this invention.

The present invention also relates to a DNA molecule encoding the humanMnSOD gene. The nucleotide sequence of the exon coding regions of onestrand of the MnSOD gene is shown in 6. The DNA encoding the MnSOD genemay be incorporated into a cloning vehicle such as a plasmid or a virus.The DNA or the cloning vehicle may be introduced into eucaryotic cellsusing known methods. Preferably, the DNA encoding the human manganesesuperoxide dismutase gene is encoded in the plasmids pMSG11-1, pMSG4 andpMSG-1b. The eucaryotic cells which are transformed are preferablymammalian cell lines such as the human HeLa cell line or the mouse Lcell line. Another aspect of this invention is the production of thehuman manganese superoxide dismutase polypeptide, analog, mutant orenzyme by growing the cells of this invention in suitable medium undersuitable conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The Sequence of human MnSOD cDNA

FIG. 1 shows the nucleotide sequence of one strand of a double-strandedDNA molecule encoding the human manganese superoxide dismutase as wellas the 198 amino acid sequence of human MnSOD corresponding to the DNAsequence. FIG. 1 also shows the nucleotide sequence of one strand of adouble stranded DNA molecule encoding a prepeptide to the mature humanMnSOD consisting of twenty-four amino acids and the amino acid sequencecorresponding to that DNA sequence. Also shown are the 5′ and 3′untranslated sequences.

FIG. 2. Construction of pMSE-4: Human MnSOD Expression Plasmid

Plasmid pMS8-4, containing MnSOD on an EcoRI (R₁) insert, was digestedto completion with NdeI and NarI restriction enzymes. The large fragmentwas isolated and ligated with a synthetic oligomer as depicted in FIG.2. The resulting plasmid, pMS8-NN contains the coding region for themature MnSOD, preceded by an ATG initiation codon. The above plasmid wasdigested with EcoRI, ends were filled in with Klenow fragment ofPolymerase I and further cleaved with NdeI. The small fragment harboringthe MnSOD gene was inserted into pSOD 13 which was treated with NdeI andStuI. pSOD-13 may be obtained as described in pending co-assigned U.S.patent application Ser. No. 644,245, filed Aug. 27, 1984 which is herebyincorporated by reference. This generated plasmid pMSE-4 containing theMnSOD coding region preceded by the cII ribosomal binding site and underthe control of λP_(L) promoter. Plasmid pMSE-4 has been deposited withthe American Type Culture Collection under ATCC Accession No. 53250.

FIG. 3. Effect of Mn⁺⁺ Concentration on the Activity of SOD Produced inE. Coli

The chart in FIG. 3 shows the correlation between the specific activityin units/mg of recombinant soluble MnSOD produced by E. coli strainA4255 containing plasmid pMSE-4 under both non-induction (32° C.) andinduction (42° C.) conditions, and the concentration of Mn⁺⁺ (parts permillion) in the growth medium.

FIG. 4. Construction of pMSΔRB4; Human MnSOD Expression Plasmid

Tet^(R) expression vector, pΔRB, was generated from pSODβ₁T-11 bycomplete digestion with EcoRI followed by partial cleavage with BamHIrestriction enzymes. pSODβ₁T-11 has been deposited with the AmericanType Culture Collection (ATCC) under Accession No. 53468. The digestedplasmid was ligated with synthetic oligomer

5′-AATTCCCGGGTCTAGATCT-3′

3′-GGGCCCAGATCTAGACTAG-5′

resulting in pΔRB containing the λP_(L) promoter.

The EcoRI fragment of MnSOD expression plasmid pMSE-4, containing cIIribosomal binding site and the complete coding sequence for the matureenzyme, was inserted into the unique EcoRI site of pΔRB. The resultingplasmid, pMSΔRB4, contains the MnSOD gene under control of λP_(L) andcII RBS and confers resistance to tetracycline.

FIG. 5. Restriction Map and Organization of Human MnSOD Gene

The thick line represents genomic DNA with the positions of the variousrestriction endonucleases. The black boxes numbered I-VI are the exons.The three open bars above represent the genomic clones which contain theMnSOD gene.

FIG. 6. Nucleotide Sequence of Human MnSOD Gene

The coding regions and adjacent nucleotides are shown; the exons (shadedareas) were identified by comparison with the cDNA clones. The initatorcodon (ATG), termination codon (TAA) and the polyadenylation signal(AATAAA) are underlined. The Spl hexanucleotide (GGGCGG) binding site isindicated by a line above the sequence. Dotted arrows represent possiblestem-loop structures; straight arrows are direct repeats. It should benoted that the numbers shown to the left of the Figure are arbitrary andwere selected only to assist in the identification of the regionsmentioned in the text. Neither the entire non-coding or the entirecoding region is shown.

FIG. 7. Exon-Intron Junctions of Human MnSOD Gene

The nucleotide sequence at the borders of all five introns are comparedwith the consensus sequences. Note that a shift of one nucleotide inintron #1 may alter either the donor or acceptor sequences.

FIG. 8. Pharmacokinetics of MnSOD After Subcutaneous Injection Into Rats

Time course of the serum levels of SOD enzymatic activity in rats aftersubcutaneous administration of 50 mg/kg of CuZn SOD (lower curve) orMnSOD (upper curve). Values are expressed as the mean and standard error(3 rats per point) of the enzymatic activity, calculated as ug/mlassuming a specific activity of 3000 units/mg.

FIG. 9. Comparison Between MnSOD and CuZn SOD

Effect of CuZn SOD and MnSOD administration on carrageenan-induced pawswelling in rats. MnSOD (50 mg/kg) was administered subcutaneously 24hours before carrageenan injection (−24 h Mn); CuZn SOD (50 mg/kg) wasadministered subcutaneously 2 hours (−2 h Cu) or 24 hours (−24 h Cu)prior to carrageenan injection. The control rats received carrageenanonly. The bars and vertical brackets represent the means±standard errors(8 rats per group) of the increase in paw volume 1, 2, 3 and 4 hoursafter carrageenan administration. The asterisks indicate the statisticalsignificance of the difference between the treated groups compared tothe control group: (*) p<0.05; (**) p<0.01; (***) p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

A double-stranded DNA molecule which includes cDNA encoding humanmanganese superoxide dismutase polypeptide or an analog or mutantthereof has been isolated from a human T-cell DNA library. Thenucleotide sequence of a double-stranded DNA molecule which encodeshuman manganese superoxide dismutase polypeptide or an analog or mutantthereof has been discovered. The sequence of one strand of DNA moleculeencoding the human manganese superoxide dismutase polypeptide or analogthereof is shown in FIG. 1 and includes nucleotides numbers 115 to 708inclusive. The sequence of one strand encoding hMnSOD analog or mutantis substantially similar to the strand encoding the hMnSOD polypeptide.The nucleotide sequence of the prepeptide of human manganese superoxidedismutase is also shown in FIG. 1. Nucleotides numbers 43 through 114inclusive code for this prepeptide.

The methods of preparing the cDNA and of determining the sequence of DNAencoding the human manganese superoxide dismutase polypeptide, analog ormutant thereof are known to those skilled in the art and are describedmore fully hereinafter. Moreover, now that the DNA sequence whichencodes the human manganese superoxide dismutase has been discovered,known synthetic methods can be employed to prepare DNA moleculescontaining portions of this sequence.

Conventional cloning vehicles such as plasmids, e.g., pBR322, viruses orbacteriophages, e.g., can be modified or engineered using known methodsso as to produce novel cloning vehicles which contain cDNA encodinghuman manganese superoxide dismutase polypeptide, or analogs or mutantsthereof. Similarly, such cloning vehicles can be modified or engineeredso that they contain DNA molecules, one strand of which includes asegment having the sequence shown in FIG. 1 for human manganesesuperoxide dismutase polypeptide or segments substantially similarthereto. The DNA molecule inserted may be made by various methodsincluding enzymatic or chemical synthesis.

The resulting cloning vehicles are chemical entities which do not occurin nature and may only be created by the modern technology commonlydescribed as recombinant DNA technology. Preferably the cloning vehicleis a plasmid, e.g. pMSE-4 or pMSΔRB4. These cloning vehicles may beintroduced in cells, either procaryotic, e.g., bacterial (Escherichiacoli, B.subtilis, etc.) or eukaryotic, e.g., yeast or mammalian, usingtechniques known to those skilled in the art, such as transformation,transfection and the like. The cells into which the cloning vehicles areintroduced will thus contain cDNA encoding human manganese superoxidedismutase polypeptide or analog or mutant thereof if the cDNA waspresent in the cloning vehicle or will contain DNA which includes astrand, all or a portion of which has the sequence for human MnSODpolypeptide shown in FIG. 1 or sequence substantially similar thereto ifsuch DNA was present in the cloning vehicle.

Escherichia coli are preferred host cells for the cloning vehicles ofthis invention. A presently preferred auxotrophic strain of E. coli isA1645 which has been deposited with the American Type Culture Collectionin Rockville, Md., U.S.A. containing plasmid pApoE-Ex2, under ATCCAccession No. 39787. All deposits with the American Type CultureCollection referred to in this application were made pursuant to theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms.

A1645 was obtained from A1637 by selection for Gal⁺ (ability to fermentgalactose) as well as loss of tetracycline resistance. It still containselements of phage λ. Its phenotype is C600 r⁻m⁺ gal⁺ thr⁺ leu⁻ lacZ⁻ bl(λcI857 Δ Hl Δ BamHl N⁺).

A1637 was obtained from C600 by inserting transposon containingtetracycline resistance gene into the galactose operon as well aselements of phage λ including those elements responsible for cIrepressor synthesis. C600 is available from the American Type CultureCollection, as ATCC Accession No. 23724.

Prototrophic strains of Escherichia coli which enable high levelpolypeptide expression even when grown in a minimal media are even morepreferred as hosts for expression of genes encoding manganese superoxidedismutase. One presently preferred prototrophic strain is A4255. StrainA4255 containing the plasmid pMSE-4 has been deposited with the AmericanType Culture Collection under ATCC Accession No. 53250.

The resulting cells into which DNA encoding human manganese superoxidedismutase polypeptide or analog or mutant thereof has been introducedmay be treated, e.g. grown or cultured as appropriate under suitableconditions known to those skilled in the art, so that the DNA directsexpression of the genetic information encoded by the DNA, e.g. directsexpression of the hMnSOD polypeptide or analog or mutant thereof, andthe cell expresses the hMnSOD polypeptide or analog or mutant thereofwhich may then be recovered.

As used throughout this specification, the term “superoxide dismutase”(SOD) means an enzyme or a polypeptide acting upon superoxide oroxygen-free radicals as receptors, or which catalyze the followingdismutation reaction:

20₂+2H⁺→O₂+H₂O₂

The term “manganese superoxide dismutase” (MnSOD) as used herein meansany superoxide dismutase molecule containing the element manganese, inany of its chemical forms.

The term “human manganese superoxide dismutase polypeptide” as usedherein means a polypeptide of 198 amino acids a portion of the aminoacid sequence of which is shown in FIG. 1; the N-terminus of thesequence is the lysine encoded by nucleotides 115-117 of FIG. 1 and theCOOH terminus of the sequence is the lysine encoded by nucleotides706-708 of FIG. 1.

The term “polypeptide manganese complex” as used herein means a moleculewhich includes a human manganese superoxide dismutase polypeptide in acomplex with manganese in any of its chemical forms and which has theenzymatic activity of naturally-occurring human manganese superoxidedismutase.

The term “human manganese superoxide dismutase” as used herein means amolecule which includes at least two human manganese superoxidedismutase polypeptides in a complex with manganese in any of itschemical forms and which has the enzymatic activity ofnaturally-occurring human manganese superoxide dismutase.

The term “human manganese superoxide dismutase polypeptide analog” asused herein means a polypeptide which includes a human manganesesuperoxide dismutase polypeptide to either or both ends of which one ormore additional amino acids are attached.

The term “polypeptide manganese complex analog” as used herein means amolecule which includes a polypeptide manganese complex, the polypeptideportion of which includes one or more additional amino acids attached toit at either or both ends.

The term “human manganese superoxide dismutase analog” as used hereinmeans a molecule that includes at least two polypeptides at least one ofwhich is human manganese superoxide dismutase polypeptide analog, in acomplex with manganese in any of its chemical forms, and which has theenzymatic activity of naturally-occurring human manganese superoxidedismutase.

The term “human manganese superoxide dismutase polypeptide mutant” asused herein means a polypeptide having an amino acid sequencesubstantially identical to that of the human manganese superoxidedismutase polypeptide but differing from it by one or more amino acids.

The term “polypeptide manganese complex mutant” means a molecule whichincludes a human manganese superoxide dismutase polypeptide mutant in acomplex with manganese in any of its chemical forms and which has theenzymatic activity of manganese superoxide dismutase.

The term “human manganese superoxide dismutase mutant” as used hereinmeans a molecule which includes at least two polypeptides at least oneof which polypeptides is a human manganese superoxide dismutasepolypeptide mutant in a complex with manganese in any of its chemicalforms and which has the enzymatic activity of naturally-occurring humanmanganese superoxide dismutase.

The mutants of hMnSOD polypeptide and hMnSOD which are included as apart of this invention may be prepared by mutating the DNA sequenceshown in FIG. 1, the N-terminus of which sequence is the lysine encodedby nucleotides 115-117 and the COOH terminus of which sequence isencoded by nucleotides 706-708.

The DNA may be mutated by methods known to those of ordinary skill inthe art, e.g. Bauer et al., Gene 37: 73-81 (1985). The mutated sequencemay be inserted into suitable expression vectors as described herein,which are introduced into cells which are then treated so that themutated DNA directs expression of the hMnSOD polypeptide mutants and thehMnSOD mutants.

The enzymatically active form of human manganese superoxide dismutase isbelieved to be a protein having at least two, and possibly four,identical subunits, each of which has approximately 198 amino acids inthe sequence shown in FIG. 1 for human manganese superoxide dismutase,the N-terminus of the sequence being the lysine encoded by nucleotides115-117 of FIG. 1 and the COOH terminus of the sequence being the lysineencoded by nucleotides 706-708 of FIG. 1.

Human MnSOD or analogs or mutants thereof may be prepared from cellsinto which DNA or cDNA encoding human manganese superoxide dismutase, orits analogs or mutants have been introduced. This human MnSOD or analogsor mutants may be used to catalyze the-dismutation or univalentreduction of the superoxide anion in the presence of protons to formhydrogen peroxide as shown in the following equation:

Veterinary and pharmaceutical compositions may also be prepared whichcontain effective amounts of hMnSOD or one or more hMnSOD analogs ormutant and a suitable carrier. Such carriers are well-known to thoseskilled in the art. The hMnSOD or analog or mutant thereof may beadministered directly or in the form of a composition to the animal orhuman subject, e.g., to treat a subject afflicted by inflammations or toreduce injury to the subject by oxygen-free radicals on reperfusionfollowing ischemia or organ transplantation. The hMnSOD or analog ormutant may also be added directly or in the form of a composition to theperfusion medium of an isolated organ, to reduce injury to an isolatedorgan by oxygen-free radicals on perfusion after excision, thusprolonging the survival period of the organ. Additionally, the hMnSOD oranalog or mutant thereof may be used to reduce neurological injury onreperfusion following ischemia and to treat bronchial pulmonarydysplasia.

A method of producing enzymatically active human manganese superoxidedismutase or an analog or mutant thereof in a bacterial cell has alsobeen discovered. The bacterial cell contains and is capable ofexpressing a DNA sequence encoding the human manganese superoxidedismutase or analog or mutant thereof. The method involves maintainingthe bacterial cell under suitable conditions and in a suitableproduction medium. The production medium is supplemented with an amountof Mn⁺⁺ so that the concentration of Mn⁺⁺ in the medium is greater thanabout 2 ppm.

The bacterial cell can be any bacterium in which a DNA sequence encodinghuman manganese superoxide dismutase has been introduced by recombinantDNA techniques. The bacterium must be capable of expressing the DNAsequence and producing the protein product. The suitable conditions andproduction medium will vary according to the species and strain ofbacterium.

The bacterial cell may contain the DNA sequence encoding the superoxidedismutase or analog in the body of a vector DNA molecule such as aplasmid. The vector or plasmid is constructed by recombinant DNAtechniques to have the sequence encoding the SOD incorporated at asuitable position in the molecule.

In a preferred embodiment of the invention the bacterial cell is anEscherichia coli cell. A preferred auxotrophic strain of E. coli isA1645. A preferred prototrophic strain of E. coli is A4255 The E. colicell of this invention contains a plasmid which encodes for humanmanganese superoxide dismutase or an analog or mutant thereof.

In a preferred embodiment of this invention, the bacterial cell containsthe plasmid pMSE-4. A method of constructing this plasmid is describedin the Description of the Figures and the plasmid itself is described inExample 2. This plasmid has been deposited with the ATCC under AccessionNo. 43250.

In another preferred embodiment of this invention, the bacterial cellcontains the plasmid pMSΔRB4. A method of constructing this plasmid isdescribed in the Description of the Figures and the plasmid itself isdescribed in Example 5. This plasmid may be constructed from pSODβ₁T-11which has been deposited with the American Type Culture Collection underAccession No. 53468.

In specific embodiments of the invention, an enzymatically active humanmanganese superoxide dismutase analog is produced by E. coli strainA4255 cell containing the plasmid p MSE-4 and by E. coli strain A4255cell containing the plasmid pMSΔRB4.

The suitable production medium for the bacterial cell can be any type ofacceptable growth medium such as casein hydrolysate or LB (Luria Broth)medium, the latter being preferred. Suitable growth conditions will varywith the strain of E. coli and the plasmid it contains, for example E.coli A4255 containing plasmid pMSE-4 is induced at 42° C. and maintainedat that temperature from about 1 to about 5 hours. The suitableconditions of temperature, time, agitation and aeration for growing theinoculum and for growing the culture to a desired density before theproduction phase as well as for maintaining the culture in theproduction period may vary and are known to those of ordinary skill inthe art.

The concentration of Mn⁺⁺ ion in the medium that is necessary to produceenzymatically active MnSOD will vary with the type of medium used.

In LB-type growth media Mn⁺⁺ concentrations of 150 ppm to 750 ppm havebeen found effective. It is preferred that in all complex types ofgrowth mediums the concentration of Mn⁺⁺ in the medium is from about 50to about 1500 ppm.

The specific ingredients of the suitable stock, culture, inoculating andproduction mediums may vary and are known to those of ordinary skill inthe art.

This invention also concerns a method of recovering human manganesesuperoxide dismutase or analog or mutant thereof from bacterial cellswhich contain the same. The cells are first treated to recover a proteinfraction containing proteins present in the cells including humanmanganese superoxide dismutase or analog or mutant thereof and then theprotein fraction is treated to recover human manganese superoxidedismutase or analog or mutant thereof.

In a preferred embodiment of the invention, the cells are first treatedto separate soluble proteins from insoluble proteins and cell walldebris and the soluble proteins are then recovered. The soluble proteinsso recovered are then treated to separate, e.g. precipitate, a fractionof the soluble proteins containing the human manganese superoxidedismutase or analog or mutant thereof and the fraction is recovered. Thefraction is then treated to separately recover the human manganesesuperoxide dismutase or analog or mutant thereof.

The following is a description of a more preferred embodiment of theinvention. First, the bacterial cells are isolated from the productionmedium and suspended in a suitable solution having a pH of about 7.0 or8.0. The cells are then disrupted and centrifuged. The resultingsupernatant is heated for a period of about 30 to 120 minutes at atemperature between approximately 55 to 65° C., preferably for 45-75minutes at 58 to 62° C. and more preferably one hour at 60° C., and thencooled to below 10° C., preferably to about 4° C. Any precipitate whichmay form during cooling is removed, e.g. by centrifugation and then thecooled supernatant is dialyzed against an appropriate buffer. Preferablythe cooled supernatant is dialyzed by ultrafiltration employing afiltration membrane smaller than 30K, most preferably 10K. Appropriatebuffers include 2 mM potassium phosphate buffer having a pH of about7.8. After or simultaneously with this dialysis the cooled supernatantmay optionally be concentrated to an appropriate volume, e.g. 0.03 ofthe supernatant's original volume has been found to be convenient. Theretentate is then eluted on an anion exchange chromatography column withan appropriate buffered solution, e.g., a solution at least 20 mMpotassium phosphate buffer having a pH of about 7.8. The fractions ofeluent containing superoxide dismutase are collected, pooled anddialyzed against about 40 mM potassium acetate, pH 5.5. The dialyzedpooled fractions are then eluted through a cation exchangechromatography column having a linear gradient of about 40 to about 200mM potassium acetate (KOAC) and a pH of 5.5. The peak fractionscontaining the superoxide dismutase are collected and pooled. Optionallythe pooled peak fractions may then be dialyzed against an appropriatesolution, e.g. water or a buffer solution of about 10 mM potassiumphosphate having a pH of about 7.8.

The invention also concerns purified, i.e. substantially free of othersubstances of human origin, human manganese superoxide dismutase oranalogs or mutants thereof produced by the methods of this invention. Inparticular, it concerns a human manganese superoxide dismutase analoghaving at least two polypeptides, at least one of which polypeptides hasthe amino acid sequence shown in FIG. 1, the N-terminus of whichsequence is the lysine encoded by nucleotides 115-117 of FIG. 1 and theCOOH terminus of which sequence is the lysine encoded by nucleotides706-708 of FIG. 1 plus an additional methionine residue at theN-terminus (Met-hMnSOD). A preferred embodiment of this inventionconcerns purified Met-hMnSOD having a specific activity of 3500units/mg.

The invention further concerns the ligation of human MnSOD genefragments taken from various plasmids to yield a complete human MnSODgene fragment which can be introduced into mammalian cells for theproduction of MnSOD. The various human MnSOD fragments isolated from theplasmids detail the nucleotide sequence of the genomic human MnSOD geneincluding coding and adjacent nucleotides as well as a restriction mapand organization of the gene.

The genomic gene commences at nucleotide 479 which is the firstnucleotide of the ATG starting codon and is underlined in FIG. 6. TheTAA termination codon for the genomic gene is at nucleotides 2022-2024.These numbers are arbitrary numbers merely to assist in identifying thenucleotide region.

A restriction map and organization of the genomic human MnSOD gene isdepicted in FIG. 5.

As noted, portions of genomic MnSOD DNA are found in each of threedifferent clones, pMSG11-1, pMSG4, and pMSG-1b also depicted in FIG. 5and the DNA from these clones was used to map the nucleotide sequence ofFIG. 6.

Also shown in FIGS. 6 and 7 are the exon and intron regions of the humanMnSOD gene.

This human MnSOD gene may be inserted into a plasmid which may in turnbe inserted into a eucaryotic cell capable of expressing the human gene.Methods for recovery and purification of the protein are alsocontemplated similar to those discussed above.

EXAMPLES

The Examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be construed to, limitits scope in any way. The Examples do not include detailed descriptionsfor conventional methods employed in the construction of vectors, theinsertion of genes encoding polypeptides into such vectors or theintroduction of the resulting plasmids into hosts. The Examples also donot include detailed description for conventional methods employed forassaying the polypeptides produced by such host vector systems ordetermining the identity of such polypeptides by activity staining ofisoelectric focusing (IEF) gels. Such methods are well-known to those orordinary skill in the art and are described in numerous publicationsincluding by way of example the following:

T. Maniatis, E. F. Fritsch and J. Sombrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1982).

J. M. McCord and I. Fridovich, J. Biol. Chem. 244:6049-55 (1969).

C. Beauchamp and I. Fridovich, Anal. Biochem. 44:276-87 (1971).

Example 1

In order to identify MnSOD cDNA clones, mixed oligomer probes weresynthesized according to the published amino acid sequence (18,19).

5′-probe - 30 mer sequence from AA₁₅-AA₂₄ (18,19)5′                           3′ TTGCATAATTTGTGCCTTAATGTGTGGTTC      T     G     T     G       G           G 3′-probe - 32 mer sequencefrom AA₁₇₉-AA₁₈₉ (18) 5′                             3′TCTGTTACGTTTTCCCAGTTTATTACGTTCCA   G  G              G  G

The 5′-probe consisting of 30 nucleotides corresponds to amino acids 15to 24 of mature MnSOD. The 3′-probe consisting of 32 nucleotidescorresponds to amino acids 179 to 189 of mature MnSOD. The 5′-probe is amixed probe consisting of 36 different sequences, as shown above. The3′-probe is a mixed probe consisting of 16 different sequences as shownabove. (When more than one nucleotide is shown at a given position, theDNA strand was synthesized with equimolar amounts of each of the shownnucleotides thus resulting in the mixed probe).

The 5′-probe was employed to screen 300,000 plaques of a T-cell cDNAlibrary cloned into the gt-10 vector. Hybridization to phage plaquereplicas immobilized on nitrocellulose filters was performed accordingto standard procedures (Maniatis et al. supra) except that thehybridization was performed at 50° C. in 8×SSC for 16 hrs. The filterswere then washed at 50° C. with 5×SSC and 0.1% SDS. Three positiveplaques were isolated and named Phi MS8, Phi MS1 and Phi MS1J.

EcoRI digests of DNA from Phi MS8 and Phi MS1 showed that they both havecDNA inserts approximately 800 bp long, which hybridize to both the 5′and 3′ oligonucleotide probes. Phi MS1J carried only 450 bp cDNA insertwhich hybridized only to the 5′ end probe.

The EcoRI inserts of the three phage clones were subcloned into theEcoRI site of pBR322 thus yielding pMS8-4, pMS1-4 and pMS1J,respectively. Restriction analysis and hybridization to the 5′ and 3′oligonucleotide probes revealed similar patterns for both pMS8-4 andpMS1-4. The following restriction map showing the 5′→3′ orientation hasbeen deduced for both plasmids.

The sequence of the cDNA insert of pMS8-4 is shown in FIG. 1. Thepredicted amino acid sequence differs from the published amino acidsequence (19) in that Glu appears instead of Gln in three (3) locations(AA 42, 88, 108) and an additional two amino acids, Gly and Trp appearbetween AA₁₂₃₋₁₂₄ Sequence analysis of pMS1-4 and pMS1J revealed thatthe three MnSOD clones were independently derived and confirmed thesedifferences from the published amino acid sequence.

The sequence upstream of the N-terminal Lysine of mature MnSOD predictsa pre-peptide sequence of 24 amino acids.

Example 2

Construction of pMSE-4: Amp^(R) Human MnSOD Expression Plasmid

The starting point for the construction of pMSE-4 is the plasmid pMS8-4which was obtained as described in Example 1. Plasmid pMS8-4, containinghuman MnSOD cDNA on an EcoRI insert, was digested to completion withNdeI and NarI restriction enzymes. The large fragment was isolated andligated with a synthetic oligomer as depicted in FIG. 2. The resultingplasmid, pMS8-NN contains the coding region for the mature MnSOD,preceded by an ATG initiation codon. The above plasmid was digested withEcoRI, ends were filled in with Klenow fragment of Polymerase I andfurther cleaved with NdeI . The small fragment containing the MnSOD genewas inserted into pSOD_(α)13 which was treated with NdeI and NdeI.pSOD_(α) 13 may be obtained as described in pending, co-assigned U.S.patent application Ser. No. 644,245, filed Aug. 27, 1984 which is herebyincorporated by reference. This generated plasmid pMSE-4 containing theMnSOD coding region preceded by the cII ribosomal binding site and underthe control of λP_(L) promoter. Plasmid pMSE-4 has been deposited withthe American Type Culture Collection under ATCC Accession No. 53250. Allmethods utilized in the above processes are essentially the same asthose described in maniatis, supra.

Example 3

Expression of the Recombinant Human MnSOD

Plasmid pMSE-4 was introduced into Escherichia coli strain A4255 usingknown methods. Then the E. coli strain 4255, containing pMSE-4, weregrown at 32° C. in Luria Broth (LB) medium containing 100 μg/ml ofampicillin until the Optical Density (OD) at 600 nm was 0.7. Inductionwas performed at 42° C. Samples taken at various time intervals wereelectrophoresed separated on sodium dodecyl sulfate-polyacrylamide gelselectrophoresis (SDS-PAGE). The gels showed increases in human MnSODlevels up to 120 minutes post-induction, at which stage the recombinantMnSOD protein comprised 27% of total cellular proteins as determined byscanning of Coomassie-blue stained gel. Sonication of samples for 90sec. in a W-375 sonicator and partitioning of proteins to soluble (s)and non-soluble (p) fractions by centrifugation at 10,000 g for 5 min.revealed that most of the recombinant MnSOD produced was non-soluble.The induced soluble protein fraction contained only slightly more SODactivity than the uninduced counterpart, as assayed by standard methods.See McCord et al., supra. Apparently a portion of the MnSOD found in thesoluble fraction is inactive. This suggested that most of the humanMnSOD produced under the conditions described in this Example is, ineffect, inactive.

Example 4

Effect of Mn⁺⁺ in Growth Media on MnSOD Solubility and Activity

The addition of Mn⁺⁺ in increasing concentrations up to 450 ppm to thegrowth media of E. coli A4255, containing pMSE-4, prior to a 2 hr.induction at 42° C. had no adverse effect on the overall yield of humanMnSOD. Analysis of sonicated protein fractions soluble (s) andnon-soluble (p) on sodium dodecyl sulfate-polyacryl-amide gelelectrophoresis (SDS-PAGE), showed increased solubilization of therecombinant protein with increased Mn⁺⁺ concentrations (Table 1). Anassay of SOD activity (see McCord et al., supra) suggests a correlationbetween increased Mn⁺⁺ concentrations in the growth media and increasedsolubility of the MnSOD with an apparent optimum at 150 ppm Mn⁺⁺concentration in the media (FIG. 3). Furthermore increased Mn⁺⁺concentrations activated previously inactive soluble enzyme. Solubleprotein fractions of induced cultures grown at these Mn⁺⁺ levels show upto 60-fold increase in SOD activity over soluble protein fractions ofnon-induced cultures grown at these Mn⁺⁺ levels. Activity staining ofisoelectric focusing (IEF) gels (see Beauchamp et al, .) revealed thatmulti forms of the recombinant MnSOD were identical to those of nativehuman liver MnSOD.

Results for human MnSOD production by E. coli A1645 containing pMSE-4were similar to those described above.

TABLE 1 Percent Percent Soluble Soluble human Mn human Mn Specific SODof SOD of Activity Total human Soluble units/mg Mn⁺⁺ MnSOD BacterialSoluble (ppm) Induced Proteins Proteins 0 30.6 7.2 30 50 72.7 15.4 241100 78.0 16.9 356 150 82.9 18.8 606 200 82.0 20.8 338 250 79.2 20.4 380300 80.8 20.3 381 450 89.2 22.4 323

Example 5

Construction of pMSΔRB4: Tet^(R) Human MnSOD Expression Plasmid

Tet^(R) expression vector, pΔRB, was generated from pSODβ₁T-11 bycomplete digestion with EcoRI followed by partial cleavage with BamHIrestriction enzymes. pSODβ₁T-11 has been deposited with the AmericanType Culture Collection under Accession No. 53468. The digested plasmidwas ligated with synthetic oligomer

5′-AATTCCOGGGTCTAGATCT-3′

3′-GGGCCCAGATCTAGACTAG-5′

resulting in pΔRB containing the λP_(L) promoter.

The EcoRI fragment of MnSOD expression plasmid pMSE-4, containing cIIribosomal binding site and the complete coding sequence for the matureenzyme, was inserted into the unique EcoRI site of pΔRB. The resultingplasmid, pMSΔRB4, contains the MnSOD gene under control of λPL and cIIRBS and confers resistance to tetracycline (FIG. 4).

Example 6

Expression of Human MnSOD from pMS RB4

Plasmid pMSΔRB4 was introduced into Escherichia coli strain A4255, usingknown methods. Cultures were grown at 32° C. in Luria Broth (LB)containing various concentrations of Mn⁺⁺, until the Optical Density(OD) at 600 nm reached 6.7. Induction was performed at 42° C. Samplestaken at various time intervals were electrophoresed on SDS-PAGE. hMnSODlevel increased with induction time up to 120 minutes, at which stage itcomprised about 15% of total cellular proteins as determined by scanningof Coomassie Blue stained gel.

The induced MnSOD was soluble, regardless of Mn⁺⁺ concentration ingrowth media. This is in contrast with observations for the Amp^(R)plasmid pMSE-4. (See Example 4.) However, maximum SOD activity andexpression level were dependent on Mn⁺⁺ supplementation (Table 2).

TABLE 2 MnSOD Expression in E. coli A4255 (pMS RB4) Specific PercentSoluble Activity hMnSOD Units/mg of Soluble Soluble Bacterial ProteinsProteins ppm Mn⁺⁺ 42° 32° 42° 0 10.9 8.0 23 50 19.8 8.0 227 100 16.0 8.0241 200 17.0 10.0 278 300 16.0 9.3 238

Example 7

Purification of Enzymatically Active Recombinant Human MnSOD

E. coli strain A4255 harboring plasmid pMS RB4 was fermented in LBsupplemented with 750 ppm Mn⁺⁺, at 32° C. to an A600 of 17.0. Inductionof human MnSOD expression was effected by a temperature shift to 42° C.for 2 hours at which stage the culture reached A600 of 43.0. Cells wereharvested by centrifugation and resuspended in 0.2 original volume in 50mM potassium phosphate buffer, pH 7.8 containing 250 mM NaCl. Bacteriawere disrupted by a double passage through Dynomill, centrifuged andcell debris were discarded. The supernatant was heated for 1 hour at 60°C., cooled to 40° C. and the cleared supernatant was concentrated to0.03 original volume and dialyzed against 2 mM potassium phosphatebuffer, pH 7.8, on a Pelicon ultra filtration unit equipped with a 10Kmembrane. The crude enzyme preparation was loaded onto a DE52 column,washed thoroughly with 2 mM potassium phosphate buffer, pH 7.8 andeluted with 20 mM potassium phosphate buffer, pH 7.8. Pooled fractionscontaining the enzyme were dialyzed against 40 mM potassium acetate, pH5.5, loaded onto a CM52 column and eluted with a linear gradient of40-200 mM potassium acetate, pH 5.5. Peak fractions containing humanMnSOD were pooled, dialyzed against H₂O, adjusted to 10 mM potassiumphosphate buffer, pH 7.8 and frozen at −20° C.

Recombinant human MnSOD obtained was more than 99% pure, with a specificactivity of about 3500 units/mg. The overall yield of the purificationprocedure was about 30% (Table 3).

Sequencing of the purified enzyme shows the presence of an additionalmethionine at the N-terminal amino acid as compared with the known humanMnSOD (19).

Analysis for metal content by atomic absorption revealed about 0.77atoms Mn per enzyme subunit. This is in accordance with published data(23).

TABLE 3 Purification of Recombinant Human* Mn-SOD Total Proteins YieldSpecific Activity Step gm gmSOD % units/mg Dynomill supernatant 100.011.9 100.0  417 60° C. supernatant 24.0 8.2 68.9 1197 Pelicon retentate20.0 7.5 63.0 1350 DE52 eluate 7.3 5.7 48.0 2732 CM52 eluate 4.2 4.235.3 3500 *values for enzyme purified from 15 L fermentation.

Example 8

Isolation and Structure of Human MnSOD Gene

Human placental DNA digested with HindIII and BamHI was fractionatedaccording to its size, hybridized with an MnSOD cDNA probe and thepositive enriched fractions were cloned in pBR322. Three distinct cloneswere identified according to their restriction and hybridizationpatterns: pMSG11-1, overlapping pMSG4, both comprising the 5′ end ofMnSOD gene and followed by the consecutive clone pMSG-1b which containsthe 3′ end of the gene. Plasmid pMSG11-1 has been deposited in the ATCCunder Accession No. 67363; plasmid pMSG4 has been deposited in the ATCCunder Accession No. 67364; and plasmid pMSG-1b has been deposited in theATCC under Accession No. 67365. FIG. 5 shows the restriction map andorganization of the MnSOD gene. The nucleotide sequence of the gene isshown in FIG. 6. The MnSOD gene spans a region of about 15 Kb andcontains six exons. The first intron interrupts the region coding forthe leader peptide while the last intron appears in the 3′ untranslatedregion downstream to the TAA termination codon.

The sequences of the donor and acceptor splice junctions at theexon-intron boundries are summarized in FIG. 7 and compared with theconsensus sequence. It should be pointed out that the first introncontains either an unusual donor sequence; GG instead of the highlyconserved GT (as depicted in FIG. 6), or an unusual acceptor sequence;GG instead of AG (if one moves the exon by one nucleotide). All otherfour introns are bound by the conserved GT . . . AG nucleotides.

The promoter region lacks TATA and/or CAT boxes. However, it is highlyrich in GC and contains eight repeats of the consensus hexanucleotidecore for binding transcription factor Spl (GGGCGG). Moreover, itincludes a series of direct repeats and possible stem-loop structures.The polyadenylation signal AATAAA appears 85 nucleotides downstream fromthe last exon (according to the known cDNA sequence). The sequence ofthe promoter region and the sequence of the coding regions and adjacentnucleotides was determined.

The MnSOD gene regions from plasmids pMSG11-1, pMSG4 and pMSG-1b can beconveniently isolated from the plasmids and ligated to one another toform the entire MnSOD gene. For example, the approximately 6 KBHindIII-partial BamHI fragment from pMSG11-1 can be ligated to theentire BamHI insert in plasmid pMSG4 followed by ligation to the entireBamHI insert from pMSG-1b. The result of this ligation would be a DNAfragment encoding the human MnSOD gene. This DNA fragment could then beintroduced into mammalian cells through known methods either directly orafter ligation to a cloning vehicle such as a plasmid or virus. Thetransformed cell line could then be used for production of MnSODpolypeptide, analog or mutant thereof, by culturing in a suitable mediumunder suitable conditions. The polypeptide so produced could then berecovered by methods similar to those set forth in Example 7. Thepolypeptide so recovered could then be used formulated and usedtherapeutically, for example, for treatment of ischemia or inflammation.

Example 9

Transcription of MnSOD in Human Cells

The human MnSOD cDNA from plasmid pMS-84 (FIG. 2) was hybridized topolyA⁺ RNA from human cell lines, human placenta, mouse WEHI-3 cells andbovine liver. Two species of human mRNA for MnSOD were identified, amajor transcript of human mRNA encoding MnSOD of about 1000 nucleotides(nt) long and a minor transcript of about 4000 nt in length. The mousemRNA for MnSOD is similar in size to the human major transcript, whereasmRNA for bovine MnSOD is about 300 nt longer. The long human transcript(4000 nucleotides) hybridizes to the fifth intron of the human MnSODgene, downstream from the exon coding for the carboxy terminus of theenzyme. This partially spliced transcript is non-tissue specific.

The proportion of both CuZn and MnSOD mRNAs in various cell lines was inthe order of 10⁻³%, as determined by hybridization of the SOD cDNAprobes to dot blots of serially diluted polyA⁺ RNA (Table IV). The MnSODmessage was most abundant in Hepatoma cells (2.5×10⁻³%) and CuZn SODtranscripts were most abundant in the T-lymphocyte line (4×10⁻³%).

TABLE IV Transcription Levels of MnSOD and CuZn SOD in Human Cell Lines% of polyA⁺ RNA Cell Line MnSOD CuZnSOD 1. PEER T-cell 0.6 × 10⁻³ 4.0 ×10⁻³ 2. 5637 Bladder Carcinoma 0.8 × 10⁻³ 1.6 × 10⁻³ 3. AlexanderHepatoma 2.5 × 10⁻³ 2.0 × 10⁻³

Example 10

Pharmacokinetic and Anti-Inflammatory Properties of Human RecombinantHuman MnSOD

Introduction

The anti-inflammatory activity of CuZn SOD has been demonstrated invarious biological models. The pharmokinetics of CuZn SOD afteradministration by various routes has also been examined, and it has beenfound to have a relatively short half-life (approximately 7 minutes wheninjected intravenously). By contrast, very little is known about thepharmacokinetics and biological activity of MnSOD. There is only onereport on the comparison of the pharmokinetic and anti-inflammatoryproperties of the CuZn and the Mn containing enzymes (Baret et al.,1984). Baret et al. claim that the half-life of MnSOD injectedintravenously is extremely long (6.45 hours). On the other hand, MnSODwas shown to be ineffective against carrageenan-induced paw inflammationin rats, while the CuZn SOD was effective in reducing inflammation. Inthe study described herein, we have compared the pharmacokineticproperties of recombinant human MnSOD with that of a recombinant humanCuZn SOD analog when given subcutaneously. Concurrently, we havecompared the anti-inflammatory activities of these two enzymes in thecarrageenan paw edema model. Unexpectedly, it was found that MnSOD wasefficacious in reducing inflammation in the rat model system.

Pharmacokinetic Studies

Rats were injected subcutaneously with 50 mg/kg body weight of eitherrecombinant human CuZn SOD analog or recombinant human MnSOD. Bloodsamples were drawn 0.5, 2, 4, 8, 24, 30 and 48 hours after injection andsuperoxide dismutase activity in the samples was determined by anenzymatic assay (Fridovich).

FIG. 8 summarizes the results. As shown, CuZn SOD analog values reacheda maximum of about 10 ug/ml after 2 hours and stayed at about that levelfor additional 6 hours, but dropped to pre-injection levels after 24hours. By contrast, MnSOD levels gradually increased to reach a maximallevel of about 70 ug/ml by 8 hours, and stayed at about this level forat least 30 hours. By 48 hours, the enzyme activity in serum dropped toabout 20 ug/ml, a value that was still well above the pre-injectionlevels.

Anti-Inflammatory Activity

The rat model of carrageenan-induced paw edema was used to assay theanti-inflammatory activity of CuZn SOD analog and MnSOD. In this model,Wistar-derived male rats (130-150g b.w.) were given a sub-plantarinjection of 0.1 ml of 0.1% w/v carrageenan into the left hind paw. Thepaw volume was measured by an Hg-displacement volumeter (a modificationof a Ugo-Basile volumeter, Camerio, Italy) before and at hourlyintervals after paw injection. Animals were divided into 4 groups (8rats/group). One group received a subcutaneous injection of 50 mg/kg ofMnSOD 24 hours before carrageenan administration (−24 h Mn; cf. FIG. 9).The second group was injected with Cu/Zn SOD analog (50 mg/kg) 24 hoursbefore the carrageenan challenge (−24 h Cu), while the third group wasinjected with 60 mg/Kg of CuZn SOD analog only 2 hours before thechallenge (−2 h Cu). The fourth group did not receive any pretreatmentand served as a control.

The results are shown in FIG. 9. As seen, the administration of CuZn SODanalog 2 hours before the induction of inflammation resulted in a 50%reduction of the swelling response. In contrast, pretreatment with theCuZn enzyme 24 hours before challenge was without effect. However, a24-hour pretreatment with MnSOD resulted in an anti-inflammatoryresponse which was similar to the effect of the 2 hours pretreatmentwith CuZn SOD analog.

Conclusions

It has been demonstrated herein that the rate of disappearance ofrecombinant human MnSOD in the rat is much lower than that of therecombinant CuZn SOD analog. This is in agreement with the previousreport of Baret et al. (1984) concerning the natural human MnSOD.However, the manganese-containing enzyme has been shown herein to beactive in vivo as an anti-inflammatory agent—an activity that isattributed to its superoxide dismutase ability. This finding issurprising in view of the report of Baret et al. (1984), which claimedthat MnSOD was not active in a similar system. The finding that MnSODremains efficacious as an anti-inflammatory drug even 24 hours afteradministration indicates that it may be used as a long-actingtherapeutic agent.

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What is claimed is:
 1. A method of treating a subject afflicted withinflammation which comprises administering to the subject an effectiveamount of a recombinant polypeptide having an amino acid sequencesubstantially identical to that of human superoxide dismutase.
 2. Amethod of reducing injury to a subject occurring upon reperfusionfollowing ischemia which comprises administering to the subject aneffective amount of a recombinant polypeptide having an amino acidsequence substantially identical to that of human manganese superoxidedismutase.
 3. A method of prolonging the survival period of an isolatedorgan which comprises adding an effective amount of a recombinantpolypeptide having an amino acid sequence substantially identical tothat of human superoxide dismutase to the perfusion medium of theisolated organ.
 4. A method of treating a subject afflicted withbronchial pulmonary dysplasia which comprises administering to thesubject an effective amount of a recombinant polypeptide having an aminoacid sequence substantially identical to that of human manganesesuperoxide dismutase.